Zebrafish has become an important organism for the study of vertebrate development due to its accessibility to forward genetics, embryonic manipulation and transgenic analysis (see, e.g. Amsterdam, A et al, Genes Dev 13, 2713-24 (1999); Long, Q et al., Development 124, 4105-11 (1997); Haffter, P et al., Development 123, 1-36 (1996); Solnica-Krezel, L et al., Development 123, 67-80 (1996)). Methods are available to perform large-scale mutagenesis screens, allowing identification of key regulatory genes in development. In addition, transparent zebrafish embryos are well suited for manipulations involving DNA or mRNA injection, cell labeling, and transplantation. Nevertheless, to fully realize the potential of this organism, tools of reverse genetics are needed. Once the scheduled zebrafish genome project is complete, targeted genetic manipulations will become even more desirable for zebrafish. Although zebrafish cell cultures exhibiting some characteristics of embryonic stem cells have been described, such short-term cell cultures do not provide adequate time for genetic manipulation such as gene knockouts (see, e.g. Ma et al., Proc Natl Acad Sci USA 98, 2461-6 (2001)).
As an alternative to embryonic stem cells, production of cloned animal using cultured cells offers the possibility of targeted genetic manipulations (see, e.g. Lai, L et al., Science 295, 1089-92 (2002); McCreath, K J et al., Nature 405, 1066-9 (2000)). Nuclear transfer has been in progress for approximately 50 years since its first demonstration in frogs (see, e.g. Briggs, R & King, T J Proc Natl Acad Sci USA 38, 455-63 (1952)). Since then, amphibians have been commonly used for studying nuclear transfer. However, subsequent reports with differentiated somatic cells did not produce individuals that could survive beyond the tadpole stage (see, e.g. Gurdon, J B Adv Exp Med Biol 62, 35-44 (1975); Wabl et al., Science 190, 1310-2 (1975); Di Berardino et al., Differentiation 50, 1-13 (1992); Orr et al., Proc Natl Acad Sci USA 83, 1369-73 (1986)). Similar attempts were made to produce cloned animals in cattle, sheep, rabbit, and pig for commercial benefits (see, e.g. Prather et al., Biol Reprod 37, 859-66 (1987); Willadsen, S M Nuclear transplantation in sheep embryos Nature 320, 63-5 (1986); Stice, S L & Robl, J M Biol Reprod 39, 657-64 (1988); Prather et al., Biol Reprod 41, 414-8 (1989)). After the birth of Dolly the sheep from differentiated cells (see, e.g. Wilmut et al., Nature 385, 810-3 (1997)), a number of successful animal cloning experiments using somatic cells have been achieved, including recent reports producing “gene-knockout” sheep and pigs by nuclear transfer from genetically manipulated somatic cells (see, e.g. Lai, L et al., Science 295, 1089-92 (2002); McCreath, K J et al., Nature 405, 1066-9 (2000)).
Nuclear transfer in fish has been studied since the 1960's. Gasaryan et al. transplanted uncultured blastula nuclei into non-enucleated and enucleated eggs of the loach and obtained feeding larvae (see, e.g. Gasaryan et al., Nature 280, 585-7 (1979)). Fish nuclei of blastula cells from different genera were transplanted into enucleated eggs to study nucleo-cytoplasmic interaction (see, e.g. Zhu, Z Y & Sun, Y H Cell Res 10, 17-27 (2000)). Recently, Wakamatsu et al. demonstrated that diploid and fertile medaka could be produced by nuclear transfer using blastula cells as donors (see, e.g. Wakamatsu, Y Proc Natl Acad Sci USA 98, 1071-6 (2001)). These findings show that nuclei prepared from fresh blastula cells can be reprogrammed in fish to support embryonic and adult development.
In existing technologies involving fish, donor cells typically non-cultured blastula cells that have very limited potential for in vitro genetic manipulation. Consequently there is a need in the art for methods that overcome the limitations associated with the existing technologies. The methods disclosed herein satisfy this need. This disclosure therefore provides a significant advancement in technologies involving the targeted genetic manipulation of fish.